SKLB1002, a Novel Potent Inhibitor of VEGF Receptor 2 Signaling, Inhibits Angiogenesis and Tumor Growth In Vivo

Abstract

Purpose: VEGF receptor 2 (VEGFR2) inhibitors, as efficient antiangiogenesis agents, have been applied in the cancer treatment. However, currently most of these anticancer drugs suffer some adverse effects. Discovery of novel VEGFR2 inhibitors as anticancer drug candidates is still needed. Experimental Design: In this investigation, we adopted a restricted de novo design method to design VEGFR2 inhibitors. We selected the most potent compound SKLB1002 and analyzed its inhibitory effects on human umbilical vein endothelial cells (HUVEC) in vitro. Tumor xenografts in zebrafish and athymic mice were used to examine the in vivo activity of SKLB1002. Results: The use of the restricted de novo design method indeed led to a new potent VEGFR2 inhibitor, SKLB1002, which could significantly inhibit HUVEC proliferation, migration, invasion, and tube formation. Western blot analysis was conducted, which indicated that SKLB1002 inhibited VEGF-induced phosphorylation of VEGFR2 kinase and the downstream protein kinases including extracellular signal-regulated kinase, focal adhesion kinase, and Src. In vivo zebrafish model experiments showed that SKLB1002 remarkably blocked the formation of intersegmental vessels in zebrafish embryos. It was further found to inhibit a new microvasculature in zebrafish embryos induced by inoculated tumor cells. Finally, compared with the solvent control, administration of 100 mg/kg/d SKLB1002 reached more than 60% inhibition against human tumor xenografts in athymic mice. The antiangiogenic effect was indicated by CD31 immunohistochemical staining and alginate-encapsulated tumor cell assay. Conclusions: Our findings suggest that SKLB1002 inhibits angiogenesis and may be a potential drug candidate in anticancer therapy. Clin Cancer Res; 17(13); 4439–50. ©2011 AACR.

Proteomics Identification of Annexin A2 as a Key Mediator in the Metastasis and Proangiogenesis of Endometrial Cells in Human Adenomyosis

“…99040201) was cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum. Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical cord veins using a standard procedure as previously described (14) and grown in EBM-2 medium with SingleQuots TM (Lonza, Walkersville, MD). HUVEC at passages 3-8 were used for all experiments.…”

“…Immunoblotting and ELISA-For immunoblotting, the whole cell lysates were prepared as described previously (14). Proteins from conditioned medium samples were precipitated by mixing 1 ml:5 ml with methanol and incubating for 1 h at Ϫ80°C, pelleted, dried, and then subjected to further immunoblotting.…”

“…Immunofluorescent Microscopy-Immunofluorescent microscopy was carried out as described previously (14). Stained sections were viewed and photographed using a fluorescence microscope (Olympus Optical Co., Hamburg, Germany).…”

Antitumor activity of placenta-derived mesenchymal stem cells producing pigment epithelium-derived factor in a mouse melanoma model

“…The Transwell migration assay was used to determine the effect of PEDF secreted from Ad-PEDF-infected PDMSCs on HUVECs and was performed as previously described (18,19). Briefly, HUVECs (2x10 4 per well) were suspended in 200 µl of the CM derived from PDMSCs, PDMSC-LacZ and PDMSC-PEDF, respectively, and seeded in the upper chamber which was coated with 50 µl Matrigel.…”

Therapeutic potential of bone marrow-derived mesenchymal stem cells producing pigment epithelium-derived factor in lung carcinoma

“…To determine whether PEDF secreted by MSCs-PEDF can inhibit HUVEC invasion, a Transwell invasion assay was performed as previously described (28). Briefly, the filter of the Transwell chamber (Millipore) was coated with 50 µl Matrigel (BD Biosciences).…”